TET1 dioxygenase is required for FOXA2-associated chromatin remodeling in pancreatic beta-cell differentiation

Existing knowledge of the role of epigenetic modifiers in pancreas development has exponentially increased. However, the function of TET dioxygenases in pancreatic endocrine specification remains obscure. We set out to tackle this issue using a human embryonic stem cell (hESC) differentiation system, in which TET1/TET2/TET3 triple knockout cells display severe defects in pancreatic β-cell specification. The integrative whole-genome analysis identifies unique cell-type-specific hypermethylated regions (hyper-DMRs) displaying reduced chromatin activity and remarkable enrichment of FOXA2, a pioneer transcription factor essential for pancreatic endoderm specification. Intriguingly, TET depletion leads to significant changes in FOXA2 binding at the pancreatic progenitor stage, in which gene loci with decreased FOXA2 binding feature low levels of active chromatin modifications and enriches for bHLH motifs. Transduction of full-length TET1 but not the TET1-catalytic-domain in TET-deficient cells effectively rescues β-cell differentiation accompanied by restoring PAX4 hypomethylation. Taking these findings together with the defective generation of functional β-cells upon TET1-inactivation, our study unveils an essential role of TET1-dependent demethylation in establishing β-cell identity. Moreover, we discover a physical interaction between TET1 and FOXA2 in endodermal lineage intermediates, which provides a mechanistic clue regarding the complex crosstalk between TET dioxygenases and pioneer transcription factors in epigenetic regulation during pancreas specification.

with a randomly selected set of methylated regions that consists of the same number/size of regions as hyper-DMRs in TET-deficient progenitors). Still, this would leave unresolved when these T2D risk variants exert their function. More informative would be to test whether hyper-DMRs in TET-deficient progenitors are enriched for T2D variants independent of hyper-DMRs in islets. 5. The authors make the argument that TET1 is the dominant TET dioxygenase relevant for pancreatic development. This claim could be bolstered by examining what percentage of TET1 binding sites overlap with the identified hypo-DHMRs and hyper-DMRs in TKO cells. It should be tested whether there is a statically significant enrichment. Furthermore, the authors should conduct analysis for TET1 binding across clusters in Figure 2b similar to what is performed for FOXA2 in Figure 2d. One would predict that TET1 binding sites in PP are enriched in the PP-specific cluster. Likewise, in Figure 3c is there a difference in TET1 binding between pancreas-specific and non-pancreatic hyper-DMRs? 6. In Figure 5f, the differences between common and PP-specific TET1 binding sites are subtle and inconsistent between different marks. Therefore, the conclusions drawn from this analysis are not entirely supported by the data. For example, proximal common binding sites appear more enriched for H3K4me3 signal than proximal PP-specific binding sites, while the opposite pattern is observed for HeK27me3 signal. The same discrepancy is observed between H3K4me1 signal and H3K27ac signal at distal binding sites. To determine whether these differences in ChIP-seq signal are significant, the authors should provide box and whisker plots and calculate p-values. They should address the observed inconsistencies. 7. The authors state that the indicated enhancer in Figure 6a is co-bound by FOXA2 and TET1. However, on the displayed genome browser track, the FOXA2 signal at the relevant enhancer is dispersed and the scale (0.46) is extremely low. This may be background signal as opposed to an actual FOXA2 peak. In all displayed genome browser snap shots, it should be indicated that highlighted regions of TF binding are identified as peaks by an unbiased peak-caller. Minor comments: -In Figure S1e, it should be clarified whether results from TKO clone 2 or 6 are displayed in the graph.
-The argument is made that gene expression changes in TKO cells are relatively specific to the PP stage. Although the authors provide convincing evidence that several key marker genes of the PP stage are dysregulated and that marker genes of earlier stages are relatively unaffected ( Figure  1d, S1e, S1f), a genome-wide analysis should be conducted to substantiate the conclusion. For example, the authors could conduct PCA analysis of control and TKO cells at each stage of differentiation.
- Figure 2b shows that many of the identified hypo-DHMRs have the strongest 5hmC signal at the DE stage in control cells. How do the authors explain that loss of hydroxymethylation at these sites does not impair DE formation? This should be discussed. -The heatmaps in Figure 2d are redundant with the information displayed in the density plots. I recommend removing the heatmaps.
-The methods section indicates that a 2D differentiation protocol was used. However, the immunostaining in Figure 4c appears to be from 3D aggregates. Can the authors explain? -In Figure 4a, expression of other pancreatic TFs (see Figure S1e) should be analyzed in addition to PAX4 to determine whether there is any specificity to PAX4. If other TFs are regulated, the conclusions about TET1 acting specifically by regulating PAX4 should be tempered. -In Figure 5d, the authors compare enriched motifs in ES-specific and PP-specific TET1 binding sites. A more direct way of performing this comparison would be to identify motifs enriched in ESspecific TET1 peaks against a background of PP-specific ChIP-seq peaks and vice versa. -line 281, "TET 1 bound more strongly"; line 282, "Higher active chromatin signals" -Language is used that infers quantitative changes, but no quantitative analysis is performed (see comment 6). -line 86, "ARX was not affected" -Figure S1e shows upregulation -line 241, "significantly inhibited" -downregulated

Reviewer #2 (Remarks to the Author):
In the present study, Wu and co-authors explore the function of TET enzymes in pancreatic specification by using a human embryonic stem cell differentiation system. They find that the loss of all three TET family members significantly impair the differentiation of pancreatic beta cells. By multiple omics analysis they identify cell type-specific hypermethylated regions with altered chromatin features. Furthermore, transduction of TET1 in TET-deficient cells effectively rescued beta cell differentiation and prevented hypermethylation. ChIP-seq mapping of TET1 showed that TET1 co-localized on a subset of FOXA2 targets featuring high levels of active chromatin modifications in pancreatic progenitors. The manuscript is clearly written and most of the conclusions are supported by reasonable experimental evidence. Although the link between transcription factor (TF) binding and DNA hydroxymethylation by TET enzymes was already well established in other publications, the question whether TET enzymes are needed for TF-mediated chromatin remodeling is less clear. Unfortunately, the authors fail to provide enough experimental evidence to support this claim. Thus, the following points need to be addressed to convincingly demonstrate that TET1 is required for FOXA2-associated chromatin remodeling in pancreatic beta cell differentiation. 1) The authors use a previously generated FOXA2 ChIP-seq profile (Lee et al. Cell Rep, 2019), which was obtained in a similar but not identical differentiation system. I see the precise mapping of FOXA2 binding sites key for elucidating the link with TET1. As FOXA2 binding is highly cell typespecific, slight differences in differentiation conditions can lead to redistribution of FOXA2 to other sites. It is also very important to consider that TET tko cell display strong differentiation defects, which are likely to result in altered FOXA2 binding and, in turn, altered chromatin accessibility. Therefore, I see it crucial to obtain FOXA2 ChIP-seq data in wild type and TET ko situations. 2) Since TET ko impairs pancreatic endoderm differentiation it is difficult to distinguish between direct and indirect effects on FOXA2 binding sites. As mentioned in (1), FOXA2 could be redistributed to other binding sites, leading to reduced accessibility on original peaks. Other effects may be in place here. For example, in different experimental systems it was shown that co-binding of transcription factors is needed to establish chromatin accessibility. In the human beta cell differentiation system, FOXA2 is likely to pair with beta cell specific transcription factors. Lack of those TFs or redistribution of them may also result in reduced chromatin accessibility on FOXA2 binding sites. To better disentangle the interplay between TET and FOXA2 it would be necessary to investigate an experimental system with minimal phenotypic and transcriptional changes upon TET ko. For example, the authors may investigate FOXA2 binding and chromatin accessibility in wildtype vs TET ko human ES cells.
3) ChIP-seq for TET1 resulted in only partial overlap with FOXA2 binding sites. It would be important to show quality controls for the specificity of the TET1 antibody.
Minor points: 4) lines 165-166 "suggesting that inhibition of TET did not alter the expression levels of pioneer TFs". Only data for FOXA and GATA are shown. 5) lanes 181-182: "consistent with a suggested connection between hypomethylation and activation of transposable elements". Are transposable elements upregulated in TET KO cells? 6) lines 192-193: "we analyzed differentially methylated regions (DMRs) by connecting at least three consecutive DMCs". What is the rational for this approch? 7) Fig 3d,e: This density plots should be accompanied by heat maps showing the signal distribution across the considered genomic regions. 8) line 200: "demethylation is primarily associated with pioneer TF binding". Did the author check for the presence of non-pioneering TFs? Here, it would be nice to decipher co-binding of TFs (related to point (2)). 9) Supplementary Fig 3h and Fig 6a: The signal scale for FOXA2 track is set to 0.87 and 0.59 respectively while in Fig 3g is set to 88. In Fig 3g the Foxa2 signal appears like a robust peak, but in the other figures, the Foxa2 track basically shows background signal (which would become obvious with using the same scale). The claim in lines 874-876 that Fig. 6a represents a FOXA2/TET1 co-bound region needs to be reconsidered. 10) Supplementary Fig 4a: one can hardly appreciate differences between these pictures. A quantification of the signals could be provided.

General Response to Reviewers:
We thank all reviewers for their positive and helpful comments. We were enthused by the reviewers' generally positive response to our work. We concur with the reviewers that the study would benefit from further insight into the interaction of TET1 with different TFs and FOXA2 occupancy in wildtype and TET-knockout cells. Since first submission of the manuscript, we conducted genome-wide analysis of FOXA2 occupancy and co-immunoprecipitation of TET1. Integrative analysis of FOXA2 distribution with chromatin state in wildtype and TET-depleted cells at the definitive endoderm and pancreatic progenitor stages has revealed informative mechanistic insight into how FOXA2 and TETs cooperatively control cell lineage decisions.
Key novel findings that have emerged from our analysis include: 1. We mapped FOXA2 occupancy by ChIP-seq in WT and TKO cells at the DE and PP stages.
We found that FOXA2 binding was dramatically changed at the PP stage but not at the DE stage in TET-deficient cells. We discover that 1) de novo FOXA2 recruitment at genomic loci with low levels of active chromatin modifications is decreased upon TET depletion, and 2) FOXA2-decreased binding sites are enriched with bHLH TFs, such as NEUROD1 and PTF1A, which fail to be induced in TET-knockout cells.
2. We identify specific interaction between TET1 and FOXA2 by co-immunoprecipitation in endodermal lineage intermediates, while co-precipitation of other endoderm proteins, such as SOX17, GATA6, and FOXA1, is not observed.
3. We show that transduction of full-length TET1 but not the TET1-catalytic-domain in TETdepleted cells effectively rescues β-cell differentiation accompanied by restoring PAX4 hypomethylation and expression.
Regarding the specificity of TET1 ChIP-seq, we were not able to verify it because the TET1 antibody (Sigma 09-872) we used for ChIP-seq was discontinued. We tried to examine other commercially available TET1 antibodies, but none of them shown specific pull-down in WT cells when compared to TET1-knockout cells. To ensure the best possible accuracy for TET1 occupancy, we decided to not include the TET1 ChIP-seq results into the revised manuscript and substantially revised our manuscript with new experimental results. Below we provide point-by-point responses to the concerns raised by each reviewer, and actions taken to address each concern.
Reviewer #1 (Remarks to the Author): In this manuscript, Wu et al. investigate the role of TET dioxygenases in pancreatic endocrine cell specification. They generated a TET1/TET2/TET3 -/-(TKO) hESC line and demonstrate that loss of TET activity leads to significant reduction in the numbers of PDX1+/NKX6.1+ pancreatic progenitor cells and a reduction in hormone+ cells, indicating a requirement for TET dioxygenases in endocrine cell specification. This defect correlates with cell type-specific decreases in hydroxymethylated regions and increases in hypermethylated regions in TKO cells. These regions also show reduced chromatin accessibility and H3K27 acetylation, suggesting a loss of active chromatin regions. The same regions are enriched for binding by FOXA2. Since FOXA2 is a pioneer TF that can contact closed chromatin, the authors propose that TET dioxygenases interact with FOXA TFs during endocrine cell specification to generate regions of open chromatin. In the second part of the study, the authors demonstrate that this phenotype is mainly driven by TET1, establishing this enzyme as the dominant TET dioxygenase during pancreatic endocrine cell development.
Since this is the first study to investigate the function of TET dioxygenases in pancreas development, the work is novel and relevant to the fields of epigenomics and cell differentiation. Overall, the experiments are thoroughly conducted and provide mostly convincing evidence to support the conclusions. However, several key points require clarification or additional evidence to make the work suitable for publication. In particular, changes in 5mhC and DNA methylation need to be better integrated and related to TET1 binding. Furthermore, the analysis of T2D variant enrichment at TETregulated sites is unconvincing.
Major comments: 6. In Figure 5f, the differences between common and PP-specific TET1 binding sites are subtle and inconsistent between different marks. Therefore, the conclusions drawn from this analysis are not entirely supported by the data. For example, proximal common binding sites appear more enriched for H3K4me3 signal than proximal PP-specific binding sites, while the opposite pattern is observed for HeK27me3 signal. The same discrepancy is observed between H3K4me1 signal and H3K27ac signal at distal binding sites. To determine whether these differences in ChIP-seq signal are significant, the authors should provide box and whisker plots and calculate p-values. They should address the observed inconsistencies. Figure 6a is co-bound by FOXA2 and TET1.

The authors state that the indicated enhancer in
However, on the displayed genome browser track, the FOXA2 signal at the relevant enhancer is dispersed and the scale (0.46) is extremely low. This may be background signal as opposed to an actual FOXA2 peak. In all displayed genome browser snap shots, it should be indicated that highlighted regions of TF binding are identified as peaks by an unbiased peak-caller.
Minor comments: -In Figure S1e, it should be clarified whether results from TKO clone 2 or 6 are displayed in the graph.
-The argument is made that gene expression changes in TKO cells are relatively specific to the PP stage. Although the authors provide convincing evidence that several key marker genes of the PP stage are dysregulated and that marker genes of earlier stages are relatively unaffected (Figure 1d, S1e, S1f), a genome-wide analysis should be conducted to substantiate the conclusion. For example, the authors could conduct PCA analysis of control and TKO cells at each stage of differentiation.
- Figure 2b shows that many of the identified hypo-DHMRs have the strongest 5hmC signal at the DE stage in control cells. How do the authors explain that loss of hydroxymethylation at these sites does not impair DE formation? This should be discussed.
-The heatmaps in Figure 2d are redundant with the information displayed in the density plots. I recommend removing the heatmaps.
-The methods section indicates that a 2D differentiation protocol was used. However, the immunostaining in Figure 4c appears to be from 3D aggregates. Can the authors explain? -In Figure 4a, expression of other pancreatic TFs (see Figure S1e) should be analyzed in addition to PAX4 to determine whether there is any specificity to PAX4. If other TFs are regulated, the conclusions about TET1 acting specifically by regulating PAX4 should be tempered.
-In Figure 5d, the authors compare enriched motifs in ES-specific and PP-specific TET1 binding sites.
A more direct way of performing this comparison would be to identify motifs enriched in ES-specific TET1 peaks against a background of PP-specific ChIP-seq peaks and vice versa.
-line 281, "TET 1 bound more strongly"; line 282, "Higher active chromatin signals" -Language is used that infers quantitative changes, but no quantitative analysis is performed (see comment 6). Response: We agree with reviewer that additional analysis on co-binding of TET and TFs will further strengthen the conclusion. To determine potential interaction between TET1 and FOXA2, we performed co-immunoprecipitation experiment. Due to the lack of proper TET1 antibody for IP, we applied an anti-FLAG antibody to pull down TET1 in the TKO-TET1FL cells which expressed a FLAG-tagged full-length TET1 gene. We have demonstrated that transduction of full-length TET1 in TET-deficient cells effectively rescues β-cell differentiation accompanied by restoring PAX4 hypomethylation and expression ( Fig. 6; Supplementary Fig. 6). It is therefore reasonable, although not ideal, to apply a FLAG antibody to pull down TET1 and examine its interaction partners in TKO-TET1FL cells. All tested endodermal TFs, including FOXA2, FOXA1, GATA6, and SOX17, were expressed at similar levels between TKO-TET1FL and TKO cells at the DE stage. Of particular interest was the observation that only FOXA2 but not FOXA1, GATA6, and SOX17 was co-precipitated with TET1 suggesting TET1 specifically interacts with FOXA2 in endodermal lineage intermediates. We show the new analysis of TET1 coimmunoprecipitation in Fig. 6c and Supplementary Fig. 6d of the revised manuscript. Figure 2d, the authors show FOXA2 signal at identified groups of hypo-DHMRs. It would be useful to integrate existing datasets for ATAC-seq, H3K4me1 and H3K27ac ChIP-seq at DE, GT, and PP stages to examine whether chromatin accessibility and enhancer activity show the same temporal patterns as FOXA2 binding in each group of hypo-DHMRs.

In
Response: As suggested by the reviewer, we integrated existing datasets for ATAC-seq (GSE114101), H3K4me1 ChIP-seq (GSE117136), and H3K27ac ChIP-seq (GSE117136) and analyzed their temporal patterns at 'differentiation-specific hypo-DHMRs' clusters identified in Fig. 3b. Consistent with correspondence between the dynamic distribution of FOXA2 and 5hmC (Fig. 3d), we found that ATAC-seq, H3K4me1, and H3K27ac signals showed concordant changes with 5hmC signals in most groups, but not as precise as the temporal patterns of FOXA2 (Figure 1; to reviewer only). Response: As suggested, we perform systematic analysis of hyper-DMRs in TKO_PP versus T2Dassociated hyper-DMRs in islets. We found that although hyper-DMRs in TKO_PP were significantly enriched for T2D-associated hyper-DMRs, less than 6% of the regions were overlapped (Figure 3; for reviewer only). It is therefore difficult to conclude whether those overlapped hyper-DMRs exert their function at developmental stages. The reviewer also suggests testing whether hyper-DMRs in TETdeficient pancreatic progenitors contributes to T2D pathogenesis. We total agree with the reviewer, yet our current study mainly focuses on the epigenetic regulation of TETs in pancreatic endocrine lineage specification. We decided to not include the contexts related to T2D in the revised manuscript and will conduct more comprehensive analysis in our following studies.  binding across clusters in Figure 2b similar to what is performed for FOXA2 in Figure 2d. One would predict that TET1 binding sites in PP are enriched in the PP-specific cluster. Likewise, in Figure 3c is there a difference in TET1 binding between pancreas-specific and non-pancreatic hyper-DMRs?
Response: Because the antibody (Sigma 09-872) we used for TET1-ChIP seq was discontinued, we were not able to perform additional experiment to verify its specificity. To ensure the best possible accuracy for TET1 occupancy, we decided to not include the TET1 ChIP-seq results in the revised manuscript.
6. In Figure 5f, the differences between common and PP-specific TET1 binding sites are subtle and inconsistent between different marks. Therefore, the conclusions drawn from this analysis are not entirely supported by the data. For example, proximal common binding sites appear more enriched for H3K4me3 signal than proximal PP-specific binding sites, while the opposite pattern is observed for H3K27me3 signal. The same discrepancy is observed between H3K4me1 signal and H3K27ac signal at distal binding sites. To determine whether these differences in ChIP-seq signal are significant, the authors should provide box and whisker plots and calculate p-values. They should address the observed inconsistencies.
Response: To ensure the best possible accuracy for TET1 occupancy, we decided to not include the TET1 ChIP-seq results in the revised manuscript.
7. The authors state that the indicated enhancer in Figure 6a is co-bound by FOXA2 and TET1. However, on the displayed genome browser track, the FOXA2 signal at the relevant enhancer is dispersed and the scale (0.46) is extremely low. This may be background signal as opposed to an actual FOXA2 peak. In all displayed genome browser snap shots, it should be indicated that highlighted regions of TF binding are identified as peaks by an unbiased peak-caller.
Response: We thank reviewer for pointing out these issues. We implied our own FOXA2 ChIP-seq data to replace the previously generated FOXA2 ChIP-seq data (GSE117136) 1 . All highlighted regions of FOXA2 binding in genome-browser views of different loci were identified as peaks by MACS2 (Fig.   2g, Supplementary Fig. 2h, Fig. 6d).
Minor comments: -In Figure S1e, it should be clarified whether results from TKO clone 2 or 6 are displayed in the graph.
Response: These results were generated from TKO clone 2. As suggested by the reviewer, we clarified it in the figure legend of Supplementary Fig.1e.
-The argument is made that gene expression changes in TKO cells are relatively specific to the PP stage.
Although the authors provide convincing evidence that several key marker genes of the PP stage are dysregulated and that marker genes of earlier stages are relatively unaffected (Figure 1d, S1e, S1f), a genome-wide analysis should be conducted to substantiate the conclusion. For example, the authors could conduct PCA analysis of control and TKO cells at each stage of differentiation.
Response: We greatly appreciate the reviewer for suggesting these experiments. We have added results of principal component analysis in Fig. 1d. We also generated RNA-seq data sets from WT and TKO cells differentiated at the GT stages and used PCA to determine which stages best distinguish between these cell populations. We believe that these results strengthen our conclusions, as they suggest that TET-deficient cells show similar transcriptome at the ES, DE, and GT stages but differed substantially at the PP stage.
- Figure 2b shows "Even 5hmC was also dynamically enriched at the DE stage (Fig. 3b), we did not find that loss of TETs Response: We agree with Reviewer 1, while Reviewer 2 asked for heat maps to show signal distribution across the considered genomic regions. We have kept both the density plots and heat maps in the revised manuscript.
-The methods section indicates that a 2D differentiation protocol was used. However, the immunostaining in Figure 4c appears to be from 3D aggregates. Can the authors explain?
Response: We apologize for omissions of the 3D differentiation in method section. We mainly used 2D differentiation strategy in this study. To perform immunofluorescence staining and FACS analysis on the same differentiated cells, we also applied 3D differentiation protocol. We have amended the corresponding method sections and included the 3D differentiation protocol (line 452-457).
-In Figure 4a, expression of other pancreatic TFs (see Figure S1e) should be analyzed in addition to PAX4 to determine whether there is any specificity to PAX4. If other TFs are regulated, the conclusions about TET1 acting specifically by regulating PAX4 should be tempered. -In Figure 5d, the authors compare enriched motifs in ES-specific and PP-specific TET1 binding sites.
A more direct way of performing this comparison would be to identify motifs enriched in ES-specific TET1 peaks against a background of PP-specific ChIP-seq peaks and vice versa.
Response: To ensure the best possible accuracy for TET1 occupancy, we decided to not include the TET1 ChIP-seq results in the revised manuscript.
-line 281, "TET 1 bound more strongly"; line 282, "Higher active chromatin signals" -Language is used that infers quantitative changes, but no quantitative analysis is performed (see comment 6).
Response: To ensure the best possible accuracy for TET1 occupancy, we decided to not include the TET1 ChIP-seq results in the revised manuscript.
-line 86, "ARX was not affected" -Figure S1e shows upregulation Response: We thank reviewer for pointing out these issues. Indeed, it is inaccurate to use "not affected". We changed to "not inhibited" (line 93).
-line 241, "significantly inhibited" -downregulated Response: We have corrected them in the main text (line 303).

Reviewer #2 (Remarks to the Author):
In the present study, Wu and co-authors explore the function of TET enzymes in pancreatic specification by using a human embryonic stem cell differentiation system. They find that the loss of all three TET family members significantly impair the differentiation of pancreatic beta cells. By binding sites key for elucidating the link with TET1. As FOXA2 binding is highly cell type-specific, slight differences in differentiation conditions can lead to redistribution of FOXA2 to other sites. It is also very important to consider that TET tko cell display strong differentiation defects, which are likely to result in altered FOXA2 binding and, in turn, altered chromatin accessibility. Therefore, I see it crucial to obtain FOXA2 ChIP-seq data in wild type and TET ko situations.
2) Since TET ko impairs pancreatic endoderm differentiation it is difficult to distinguish between direct and indirect effects on FOXA2 binding sites. As mentioned in (1)

Response to Reviewer #2
1) The authors use a previously generated FOXA2 ChIP-seq profile (Lee et al. Cell Rep, 2019), which was obtained in a similar but not identical differentiation system. I see the precise mapping of FOXA2 binding sites key for elucidating the link with TET1. As FOXA2 binding is highly cell type-specific, slight differences in differentiation conditions can lead to redistribution of FOXA2 to other sites. It is also very important to consider that TET tko cell display strong differentiation defects, which are likely to result in altered FOXA2 binding and, in turn, altered chromatin accessibility. Therefore, I see it crucial to obtain FOXA2 ChIP-seq data in wild type and TET ko situations.
Response: We greatly appreciate the reviewer for suggesting these experiments. We performed FOXA2 ChIP-seq in both WT and TKO cells and added the new analysis of FOXA2 occupancy in Fig. 1i, Fig.   3d, Fig. 4, and Supplementary Fig. 4 of the revised manuscript. Consistent with previous reports 1,7 , FOXA2 was primarily located at non-promotor regions (Supplementary Fig. 4a) implicating that FOXA2 is mainly involved in transcription regulation through distal regulatory elements. FOXA2 binding at each stage of differentiation in WT cells showed similar distribution as the previously published FOXA2 ChIP-seq results (revised Fig. 3d versus original Fig. 2d).
Original Fig. 2d Revised Fig. 3d 2) Since TET ko impairs pancreatic endoderm differentiation it is difficult to distinguish between direct and indirect effects on FOXA2 binding sites. As mentioned in (1) Fig. 4 and Supplementary Fig. 4.
We found that upon TET depletion, 99.5% FOXA2 binding sites did not show changes at the DE stage ( Fig. 4a), while FOXA2 occupancy was dramatically changed at the PP stage, in which 10% and 6% of FOXA2 target sites showed reduced and greater FOXA2 binding, respectively (Fig. 4a,   Supplementary Fig. 4b).
Since DNA methylation and hydroxymethylation status were significantly altered in TET-deficient cells (Fig. 2a, Supplementary Fig. 3c), we wondered whether increases in methylation or decreases in hydroxymethylation contributed to differential recruitment of FOXA2 in TKO_PP cells. We first assessed changes in FOXA2 binding in WT_PP versus TKO_PP cells at the hyper-DMRs or hypo-DHMRs identified in TKO_PP cells. We found that ~75% regions showed no changes in FOXA2 binding (Fig. 4b).
We then tested if only hypermethylated or hypohydroxymethylated FOXA2-bound sites would be affected. We found that similar proportions of FOXA2 target sites displayed reduced/greater of FOXA2 binding regardless of the presence or absence of hypermethylation (Fig. 4c). Similarly, hypo-hydroxymethylated FOXA2-bound sites did not show a preference in gain/loss of FOXA2 binding compared to iso-hydroxymethylated FOXA2-bound sites (Fig. 4d). Consistent with the ability of pioneer TFs to engage with inaccessible chromatin, our results strongly demonstrate that DNA methylation/hydroxymethylation states do not interfere in FOXA2 binding.
To comprehensively characterize temporal patterns of FOXA2 recruitment at the FOXA2-decreased binding sites, we clustered FOXA2 binding signal in WT cells at the DE, GT, and PP stages. Three distinct patterns of FOXA2 occupancy were observed. Cluster I regions (149) were bound by FOXA2 from DE to PP stage, cluster II regions (237) were FOXA2-bound at GT and PP stages, while the most predominant group, cluster III (1,888), displayed de novo FOXA2 occupancy in pancreatic progenitors ( Fig. 4e). We then analyzed the annotations of nearby genes with Genomic Regions Enrichment of Annotations Tool (GREAT) and found that cluster III regions were enriched for terms of neuron and endocrine pancreas development (Fig. 4f). Further examination of ATAC-seq, H3K27ac, and 5mC signals in three FOXA2-decreased clusters revealed high levels of DNA methylation accompanied by low levels of enhancer activity and chromatin accessibility in cluster III regions (Supplementary Fig.   4c). Taken together, our data imply that a subset of FOXA2 transiently binds to differentiation stagespecific genomic loci with low levels of active chromatin modifications at which additional lineagespecific TFs may be required to facilitate FOXA2 binding and subsequent chromatin remodeling.
To identify potential TFs associated with differential FOXA2 binding we determined unique DNA binding motifs within FOXA2-decreased, increased and stable sites. Interestingly, loci lost FOXA2 binding, particularly in cluster III, were mostly enriched for basic-helix-loop-helix (bHLH) motifs such as the pancreatic endocrine cell fate determinant NEUROD1 8 (Fig. 4g, Supplementary Fig. 4d). In contrast, regions gained FOXA2 were mostly enriched with motifs of TEAD and GATA family members, and FOXA2-stable sites most prominently feature forkhead and CTCF motifs (Fig. 4g).
Notably, expression of the bHLH TFs including NEUROD1, PTF1A, and ASCL1 failed to be induced in TET-knockout cells (Supplementary Fig. 4e) which likely results in a decrease of FOXA2 binding at genomic loci primarily associated with these TFs. In summary, these data suggest that recruitment of FOXA2 to differentiation stage-specific sites is genetically and epigenetically primed with the cooperation of additional lineage-specific TFs.
3) ChIP-seq for TET1 resulted in only partial overlap with FOXA2 binding sites. It would be important to show quality controls for the specificity of the TET1 antibody.
Response: Because the antibody (Sigma 09-872) we used for TET1-ChIP seq was discontinued, we were not able to perform additional experiment to verify its specificity. We tried to use several other commercially available TET1 antibodies to perform ChIP-qPCR. However, we found similar amplification signals between WT and TET1-knockout cells at TET1 binding sites identified from previous study 2 . To ensure the best possible accuracy for TET1 occupancy, we decided to not include the TET1 ChIP-seq results in the revised manuscript.
Minor points: 4) lines 165-166 "suggesting that inhibition of TET did not alter the expression levels of pioneer TFs". Only data for FOXA and GATA are shown.
Response: We appreciate Reviewer 2's insightful comment. FOXAs and GATAs are the known pioneer factors critical for pancreas development. They begin to express at the endoderm stage and remain high expression levels through differentiation. As suggested, we examined other pioneer TFs, such as PBX1 and TLEs, and found that their expression levels were similar between WT and TKO cells (Figure 4; for reviewer only). Response: We thank the reviewer for this comment and fully agree that association of differentiationspecific demethylation with the occupancy of pioneer TFs versus non-pioneer TFs is of an interesting point. As suggested by the reviewer, we determined enrichment of PDX1, SOX9, and HNF6 at hyper-DMRs, respectively, by integrating their ChIP-seq data generated from hESC-derived pancreatic progenitors 12 (PDX1, SOX9 and, HNF6 are non-pioneer TFs robustly expressed at the pancreatic progenitor stage). The new analysis of non-pioneer TFs was shown in Fig. 2d of the revised manuscript.
We found that lineage-specific TFs PDX1, SOX9, and HNF6 were also enriched at differentiationspecific hyper-DMRs but to a lesser extent than pioneer TFs FOXA2, GATA4, and GATA6. Taken together, we found a significant portion of hyper-DMRs was distributed in a differentiation-specific manner, in which they were enriched for both pioneer and lineage-specific TFs and showed remarkable decreases in chromatin activity upon TET inactivation (Fig 2e). Given that pioneer TFs are core components of the transcriptional complexes at cis-regulatory elements during differentiation, our data suggest that TETs are essential for enhancers activation and subsequent incorporation of lineagespecific TFs.
A similar question regarding specific interaction of TFs with TET1 was brought up by reviewer #1 (major comment 1), and we thank both reviewers for raising this important issue. To determine potential interaction between TET1 and TFs, we performed co-immunoprecipitation experiment. Due to the lack of proper TET1 antibody for IP, we applied an anti-FLAG antibody to pull down TET1 in the TKO-TET1FL cells which expressed a FLAG-tagged full-length TET1 gene. We have demonstrated that transduction of full-length TET1 in TET-deficient cells effectively rescues β-cell differentiation ( Fig.   6; Supplementary Fig. 6). It is therefore reasonable, although not ideal, to apply a FLAG antibody to pull down TET1 and examine its interaction partners in TKO-TET1FL cells. Of particular interest was the observation that only FOXA2 but not FOXA1, GATA6, and SOX17 (non-pioneer TF) was coprecipitated with TET1 (Fig. 6c, Supplementary Fig. 6d) suggesting TET1 specifically interacts with FOXA2 in endodermal lineage intermediates. Response: We thank reviewer for pointing out these issues. We replaced the previously generated FOXA2 ChIP-seq data (GSE117136) 1 with our own FOXA2 ChIP-seq data. All highlighted regions of FOXA2 binding in genome-browser views of different loci were identified as peaks by MACS2 ( Fig.   2g; Fig. 6d; Supplementary Fig. 2h).
10) Supplementary Fig 4a: one can hardly appreciate differences between these pictures. A quantification of the signals could be provided.
Response: As suggested, we performed FACS analysis and quantification of the percentage of PDX1 + and NKX6.1 + in each TET-knockout lines. Almost all mutant lines showed an ability to induce expression of PDX1 and NKX6.1 to the levels comparable to wildtype (Supplementary Fig. 5a, b). Response: We apologize for the mistake. We have carefully edited and proofread the revised manuscript to ensure that references were correctly added.

11) Fig
13) ChIP-seq protocol described in the methods would need more details.
Response: As requested, we included a detail ChIP-seq protocol in the method sections.
Because the IP validated antibodies for GATA6 and FOXA1 were on backorder, we were not able to perform reverse co-IPs on GATA6 and FOXA1. We agree with the reviewer that reverse IPs from the other TFs are needed to explore the specificity of TET1 interaction, yet our current study mainly focuses on the circuit of FOXA2 and TET1. We decided not to include the contexts related to the interaction of TET1 and GATA6/FOXA1/SOX17 in the revised manuscript and will conduct a more comprehensive analysis in our following studies. Consistent with prior reports that Tet-mediated DNA demethylation mainly occurs at many distally located enhancers 2 , we found that TET deficiency-induced DNA hypermethylation occurred at enhancers independent of FOXA2 (Figure 1a; to reviewer only). Although TET-mediated 5hmC enriched at both FOXA2-bound and unbound enhancers, higher levels of 5hmC were found at the FOXA2-bound enhancers in pancreatic progenitors (Figure 1b; to reviewer only). We, therefore, speculate that TET1 proteins co-localize to FOXA2-bound and -unbound enhancers at different levels.
As suggested, we performed Flag ChIP-qPCR using TKO-TET1FL cells differentiated at the PP stage.
We designed various pairs of primers to detect the enrichment of TET1 at FOXA2-bound (FLAG_P1, _P2, _P3, and _P4) and -unbound (FLAG_P5, _P6, _P7, and _P8) enhancers identified in WT_PP cells (Figure 1c; to reviewer only). We found that TET1_flag proteins were enriched at slightly higher levels at enhancers displaying FOXA2 deposition (Figure 1c; to reviewer only). However, ectopic the TET1_flag proteins were constitutively expressed throughout differentiation in TKO-TET1FL cells in which TET2 and TET3 were not expressed. It is therefore difficult to conclude whether the preferential enrichment of TET1 at FOXA2-bound enhancers occurs in cells expressed endogenous TET1, TET2, and TET3. To explore the precise distribution of TETs, a more comprehensive experiment using various loci-specific (TET1/TET2/TET3) tag knock-in cell lines coupled with ChIP-seq analysis will be conducted in our following studies.
To further elucidate the FOXA2-TET1 axis, we have performed additional analyses of local chromatin landscape at FOXA2-decreased and -increased sites. Analysis of chromatin accessibility, H3K27ac, and H3K4me1 signal intensity revealed more active and open chromatin at FOXA2-increased sites compared with FOXA2-decreased sites in PP (Supplementary Fig. 4c). Although DNA hypermethylation was found in both groups upon TET depletion (Supplementary Fig. 4d), H3K27ac, H3K4me1, and ATAC signals were significantly lost at the FOXA2-decreased but not -increased sites (Supplementary Fig. 4c). Together with our data showing the most predominant FOXA2-decreased regions (cluster III) displayed de novo FOXA2 occupancy in pancreatic progenitors (Fig. 4e), we believe that these results strengthen our conclusions, as they suggest that TET-mediated hypomethylation at de novo FOXA2 binding loci provides an integration hub to fine-tune cell fatespecific chromatin activation after pancreas induction.
Taken together, our analyses revealed that 1) changes in 5hmC mirror the dynamic binding of FOXA2 in cells differentiated from hESCs through defined lineage intermediates toward pancreatic endocrine fate (Fig. 2), 2) upon TET depletion chromatin activity was markedly decreased at hypohydroxymethylated regions associated with de novo FOXA2 recruitment ( Fig. 2d; Fig. 4), and 3) FOXA2 physically interacted with TET1 (Fig. 6c). Moreover, we found that de novo FOXA2-binding sites harbor motifs for bHLH TFs, such as NEUROD1 and PTF1A, which fail to be induced in TETdeficient cells, implying a subset of FOXA2 recruitment-associated chromatin activation requires lineage-specific TFs. We, therefore, postulate that FOXA2 favors TET1 deposition to facilitate local chromatin remodeling after pancreatic lineage induction, while de novo recruitment of FOXA2/TET1 provides a safeguard against broad gene expression and fine-tune high threshold cell type-specific gene expression in the pancreatic progenitor domain. Consistent with this possibility, it has been recently shown that secondary recruitment of FOXA1/2 by lineage-specific TFs to organ-specific enhancers is necessary for chromatin activation and helps establish cell type-specific gene expression within the organ progenitor domain 3 .